The rod cell is highly polarized and compartmentalized in both morphology and function. A number of genetic disorders affecting the retina are manifested in the trafficking of the visual pigment rhodopsin and the morphogenesis and/or renewal of the rod outer segment (ROS). Our previous studies showed that rhodopsin's C-terminus is a hot spot for mutations associated with retinitis pigmentosa (RP) and it contains the addressing code for its ROS delivery. We also showed that cytoplasmic dynein-based motility is essential for the directional transport of rhodopsin within the inner segment of rod. In this application, we propose to investigate the machinery and mechanisms that move rhodopsin within other distinct cellular domains of rod and their involvement in ROS formation and maintenance. In Aim1, we propose to identify molecules that capture, retain, navigate, and/or fuse rhodopsin-laden vesicles onto apical surfaces. This study will use the model of polarized MDCK epithelial cells, which have proven useful for studying the vectorial transport of rhodopsin. Aim 2 will study the physiological relevance of the interaction between rhodopsin and the FYVE domain-containing protein SARA, a novel rhodopsin C-terminus interacting protein. It has been shown that the FYVEdomain specifically binds phosphatidylinositiol-3'-phosphate, and SARA is involved in protein trafficking and membrane fusion. Our ultrastructural analysis reveals a unique distribution of SARA on the vesicles/tubules near nascent disc membranes in the proximal portion of ROS axoneme. We plan to test how the perturbation of SARA in vivo might affect rhodopsin targeting and disc morphogenesis by employing transfected rodent retinas as a model system. Conversely, the phenotypes of rods transfected with RP mutant rhodopsins that fail to bind to SARA will also be examined. In Aim3, we will identify the cellular components involved in the SARA-mediated membrane fusion pathwayin the ROS and test their importance in rhodopsin targeting and disc biogenesis in vivo. Successful achievement of the proposed specific aims will significantly further our insights into the molecular basis of the genesis and maintenance of the polarity of visual cells. These topics are a central interest in cell biology and vision research. Finally, these studies are highly relevant for our understanding of the etiology of various degenerative retinal diseases and have important implications for future rational therapies for the diseased retina.